1. Field of the Invention
This invention relates to a process for the transformation of Yarrowia lipolytica to improve its utilization for industrial purposes; to vectors and subclones thereof useful therefor, especially to vectors which replicate autonomously in Escherichia coli and integrate but do not replicate autonomously in Yarrowia lipolytica; to transformants of E. coli and Y. lipolytica containing said vectors and to their use for producing proteins.
2. Description of the Prior Art
Major emphasis in molecular cloning has been directed to prokaryotes, particularly E. coli, and more recently Bacillus subtilis, as host organisms. E. coli, despite its extensive use as host organism for the cloning and expression of heterologous DNA, is known to pose certain problems, such as the possibility of contamination of the protein produced with a toxic substance and the possible need to destroy the cells to recover the protein. This latter problem may not only aggravate the contamination problem, but may also tend to degrade the protein product and to complicate the procedures for recovering the protein.
In view of the cited difficulties, particularly toxin production, B. subtilis has been turned to as an alternative host organism since it does not produce toxins. However, as host organism, B. subtilis is subject to certain limitations; instability of transformed strains resulting in loss of heterologous DNA, and the frequent reduction in the ability of the entering DNA to coexist with the host DNA.
In recognition of the above-mentioned difficulties with prokaryotes as host organisms, attention shifted to eukaryotes, and specifically yeasts, as host organisms. Yeasts are of industrial importance, are non-toxic and can be grown to very high densities. Some species are well analyzed genetically and some species can secrete proteins.
The first transformation of a yeast, Saccharomyces cerevisiae, was reported by Hinnen et al., Proc. Natl. Acad. Sci., 75, 1929-1933 (1978) who demonstrated that a cloned segment of yeast DNA encoding the LEU2 locus could transform a nonreverting leu2.sup.- mutant of yeast to a LEU+ phenotype. This transformation was shown to result from integration into the chromosome of the plasmid containing the LEU2 segment of DNA. The integration involved recombination between homologous segments of DNA.
Hinnen et al. in "Overproduction of Microbial Products", edited by Krumphanzl et al., Academic Press, N.Y., Ch. 30, 1982, present a review of yeast transformation procedures. Typical of such procedures is that described by Ratzkin et al., Proc. Natl. Acad. Sci. 74, 487-491 (1977) in their cloning of yeast (Saccharomyces cerevisiae) LEU2 by complementation of an E. coli leuB mutation.
Sjostak et al., Plasmid 2, 536-554 (1979) describe the construction of plasmids containing the LEU2 gene of yeast and fragments of rDNA and the integration of the plasmids into the rDNA locus following yeast transformation. The basic thrust of their investigation involved the integration of a genetic marker, the LEU2 gene, inserted into the rDNA locus, as a genetic marker for mapping the rDNA. One of the reported plasmids, pSZ20, containing the BglII-B fragment of rDNA and the SalI-XhoI LEU2 fragment, is identified as a useful vector for cloning fragments of yeast DNA in yeast.
Orr-Weaver et al., Proc. Natl. Acad. Sci., 78, 6354-6358 (1981) demonstrated high frequency integration of linear plasmids derived from pBR322, all of which are nonreplicating in yeast and transform only by integration, in Saccharomyces cerevisiae when the plasmids are cut within DNA sequences homologous to the yeast chromosome.
Yarrowia lipolytica, an industrially important species of yeast, is used to produce citric acid and single cell protein. It can also be used to produce erythritol, mannitol and isopropylmalic acid. It is of special interest and value because of its ability to secrete proteins (alkaline protease, acid protease and RNAse) into its growth medium, thus permitting recovery of said proteins without the need of disrupting the producing cells. However, Y. lipolytica suffers from certain inherent deficiences, such as its limited spectrum of utilizable carbon sources. The overall value of Y. lipolytica could be increased by eliminating such deficiency as, for example, by introducing correcting DNA from another species.